In situ gene expression and ecophysiology of thermophilic Cyanobacteria

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Sheila Ingemann Jensen

Photosynthetic microbial mats in extreme light exposed environments, such as hypersaline lakes and hot springs, present ideal model systems for studying the ecology of microbial communities and the ecophysiology of particular types of microorganisms in their natural environment. Most microbial mats are dominated by prokaryotes and are characterized by the presence of steep gradients of physical and chemical variables that are closely coupled to the microbial activity and diel changes in e.g. light, temperature or salinity.

The microbial mats developing in the effluent channels of Mushroom spring, Yellowstone National Park (USA), have together with similar communities in another alkaline spring (Octopus spring) nearby, been the subject of microbial and biogeochemical investigations for >40 years. Recently, the genomes of two prominent cyanobacteria inhabiting these mats (Synechococcus OSB’ and Synechococcus OS-A) were sequenced, and the genomes of various anoxygenic phototrophs from these mats are also available. Together with emerging information from a recent large scale metagenomics project, these microbial ecosystems are ideal for investigating links between microbial diversity and activity, metabolic controls and interactions using biogeochemical and molecular tools.

This thesis builds on the extensive genomic information available from Mushroom and Octopus Springs enabling new insights into the in situ physiology and regulatory factors that might influence processes in situ. The availability of an axenic culture in the form of Synechococcus OS-B’, has furthermore enabled comparative studies of its performance in the natural environment with its performance in various controlled culture experiments.

In the thesis introduction, I will introduce the hot spring microbial mat I have worked with, as well as give a brief introduction to the cellular physiological aspects in cyanobacteria that I have been working with in this thesis. The introduction is followed by 2 published manuscripts and 2 manuscripts with research in progress.

In Manuscript 1 (published in ISME Journal, 2008, 2: 364-378), the expression patterns of various functional genes (with an emphasis on nif genes involved in N2-fixation), the protein levels of nitrogenase (NifH), the N2-fixation activity, as well as microsensor based measurements on O2 availability, production and consumption were investigated in situ over the entire diel cycle. Interestingly, it was found that while the nif genes are expressed, and nitrogenase is synthesized once the mat gets anoxic in the early evening, the largest N2-fixation activity occurs as a burst during dim light in the early morning, albeit protein levels remained high over the entire course of the night. These results point to complex regulations, where the expression levels are controlled by O2, light and protein levels, whereas the N2-fixation activity is highly dependent upon the energetic levels of the cells. It thus demonstrates an uncoupling of transcriptional activity, protein presence and actual maximal physiological activity.

In Manuscript 2 (published in ISME Journal, online publication 26 August 2010; doi: 10.1038/ismej.2010.131), the expression patterns of genes involved in photosynthesis, carbon concentrating mechanisms (CCM) and detoxification of reactive oxygen species (ROS) were investigated along with microenvironmental measurements of O2 and pH throughout the diel cycle. This is the very first study of in situ gene transcription related to CCM and ROS detoxification along with microenvironmental measurements in a microbial mat. Different transcriptional patterns were found, and the results are discussed with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle. Some transcriptional patterns could be clearly correlated to environmental changes, but due to the complexity of inter-correlated changes, they could mainly be explained by factors already investigated in cyanobacterial model organisms. This underscores the necessity of controlled culture studies. However, the inter-correlation of various endogenous and exogenous cues experienced by the cells in their natural environment also underlines the importance to integrate key environmental features in such culture studies.

In Manuscript 3, the growth and light responses of Synechococcus OS-B’ in axenic culture were investigated as a function of increasing irradiance. It is shown that high photosynthetic active radiation (PAR; 400-700 nm; 1600 µmol photons m-2 s-1) per se is not a limiting growth factor in their natural environment, but light can become a growth limiting factor for Synechococcus OS-B’ at irradiances of <200 µmol photons m-2 s-1. Cells exposed to high irradiance = 400 µmol photons m-2 s-1 showed an increased OD750:cell number ratio correlating with an observed elongation of the cells. Transcript levels of most investigated genes showed no apparent difference after >7 days acclimation to each growth irradiance. However, a gene involved in septum formation during cell division (ftsZ) exhibited decreased transcript levels in cells exposed to high irradiances. High light tolerance under replenished nutrient conditions in Synechococcus OS-B’ may thus be partially achieved by inhibition of cell division, resulting in elongated cells with a decreased pigmentation level pr. cell mass. Such inhibition may to some extent be under transcriptional control.

In Manuscript 4 we discuss the limitations of 1) in situ gene expression studies, where a mosaic of inter-correlated changes, makes it more or less impossible to decipher specific regulatory mechanisms, but do provide an answer to “how” the cells respond in their natural environment, and 2) laboratory approaches for studying transcriptional regulation in cyanobacterial cultures, i.e. continuous light vs. a dark to light period. We used in situ data from the microbial mat, where Synechococcus OS-B’ is a predominant cyanobacterium, to design environmentally relevant but yet controlled culture experiments in order to i) come closer to an understanding of cyanobacterial cell division, a parameter which is largely unknown in the microbial mats, and ii) try and decipher environmental parameters regulating transcriptional patterns observed in situ. Our laboratory studies have thus far indicated, that cells exposed to aerobic dark conditions divide both during light and darkness, whereas cell division appears to be inhibited when they are exposed to anoxic conditions in the dark, i.e. a condition more similar to the in situ reality of the cyanobacteria in the mat at nighttime.

Halts in cell division in the cultures appear to be somewhat correlated with a decreasing abundance of ftsZ gene transcripts. This is interesting, as in situ transcriptional analysis of the ftsZ gene in the microbial mat shows a distinct diel transcription pattern that is partially uncoupled from the transcription pattern of the hypothetical chromosomal replication initiator gene dnaA. The inhibition of cell division during the anaerobic dark conditions resulted in larger cells at the end of the dark period and during the following low light period. Further analysis of extracted RNA samples from the cultures and mat samples will likely provide further clues into the effect of anaerobic dark conditions on the transcription of a variety of genes.
Udgivelses stedBiologisk Institut
ForlagMuseum Tusculanum
StatusUdgivet - 2010

Bibliografisk note

Vejleder: Michael Kühl

ID: 32897268